Aromatic Ring-hydroxylating Dioxygenases Research Articles

Bacteria degrading both n-alkanes and aromatic hydrocarbons are prevalent in soils

This study was undertaken to determine the distribution of soil bacteria capable of utilizing both n-alkanes and aromatic hydrocarbons. These microorganisms have not been comprehensively investigated so far. Ten contaminated (4046–43,861 mg of total petroleum hydrocarbons (TPH) kg−1 of dry weight of soil) and five unpolluted (320–2754 mg TPH kg−1 of dry weight of soil) soil samples from temperate, arid, and Alpine soils were subjected to isolation of degraders with extended preferences and shotgun metagenomic sequencing (selected samples). The applied approach allowed to reveal that (a) these bacteria can be isolated from pristine and polluted soils, and (b) the distribution of alkane monooxygenase (alkB) and aromatic ring hydroxylating dioxygenases (ARHDs) encoding genes is not associated with the contamination presence. Some alkB and ARHD genes shared the same taxonomic affiliation; they were most often linked with the Rhodococcus, Pseudomonas, and Mycolicibacterium genera. Moreover, these taxa together with the Paeniglutamicibacter genus constituted the most numerous groups among 132 culturable strains growing in the presence of both n-alkanes and aromatic hydrocarbons. All those results indicate (a) the prevalence of the hydrocarbon degraders with extended preferences and (b) the potential of uncontaminated soil as a source of hydrocarbon degraders applied for bioremediation purposes.

Characterization of three rapidly growing novel Mycobacterium species with significant polycyclic aromatic hydrocarbon bioremediation potential.

Mycobacterium species exhibit high bioremediation potential for the degradation of polycyclic aromatic hydrocarbons (PAHs) that are significant environmental pollutants. In this study, three Gram-positive, rapidly growing strains (YC-RL4T, MB418T, and HX176T) were isolated from petroleum-contaminated soils and were classified as Mycobacterium within the family Mycobacteriaceae. Genomic average nucleotide identity (ANI; < 95%) and digital DNA-DNA hybridization (dDDH; < 70%) values relative to other Mycobacterium spp. indicated that the strains represented novel species. The morphological, physiological, and chemotaxonomic characteristics of the isolates also supported their affiliation with Mycobacterium and their delineation as novel species. The strains were identified as Mycobacterium adipatum sp. nov. (type strain YC-RL4T = CPCC 205684T = CGMCC 1.62027T), Mycobacterium deserti sp. nov. (type strain MB418T = CPCC 205710T = KCTC 49782T), and Mycobacterium hippophais sp. nov. (type strain HX176T = CPCC 205372T = KCTC 49413T). Genes encoding enzymes involved in PAH degradation and metal resistance were present in the genomes of all three strains. Specifically, genes encoding alpha subunits of aromatic ring-hydroxylating dioxygenases were encoded by the genomes. The genes were also identified as core genes in a pangenomic analysis of the three strains along with 70 phylogenetically related mycobacterial strains that were previously classified as Mycolicibacterium. Notably, strain YC-RL4T could not only utilize phthalates as their sole carbon source for growth, but also convert di-(2-ethylhexyl) phthalate into phthalic acid. These results indicated that strains YC-RL4T, MB418T, and HX176T were important resources with significant bioremediation potential in soils contaminated by PAHs and heavy metals.

Catalytic resilience of multicomponent aromatic ring-hydroxylating dioxygenases in Pseudomonas for degradation of polycyclic aromatic hydrocarbons

Hydrophobic organic compounds, either natural or introduced through anthropogenic activities, pose a serious threat to all spheres of life, including humankind. These hydrophobic compounds are recalcitrant and difficult to degrade by the microbial system; however, microbes have also evolved their metabolic and degradative potential. Pseudomonas species have been reported to have a multipotential role in the biodegradation of aromatic hydrocarbons through aromatic ring-hydroxylating dioxygenases (ARHDs). The structural complexity of different hydrophobic substrates and their chemically inert nature demands the explicit role of evolutionary conserved multicomponent enzyme ARHDs. These enzymes catalyze ring activation and subsequent oxidation by adding two molecular oxygen atoms onto the vicinal carbon of the aromatic nucleus. This critical metabolic step in the aerobic mode of degradation of polycyclic aromatic hydrocarbons (PAHs) catalyzed by ARHDs can also be explored through protein molecular docking studies. Protein data analysis enables an understanding of molecular processes and monitoring complex biodegradation reactions. This review summarizes the molecular characterization of five ARHDs from Pseudomonas species already reported for PAH degradation. Homology modeling for the amino acid sequences encoding the catalytic α-subunit of ARHDs and their docking analyses with PAHs suggested that the enzyme active sites show flexibility around the catalytic pocket for binding of low molecular weight (LMW) and high molecular weight (HMW) PAH substrates (naphthalene, phenanthrene, pyrene, benzo[α]pyrene). The alpha subunit harbours variable catalytic pockets and broader channels, allowing relaxed enzyme specificity toward PAHs. ARHD's ability to accommodate different LMW and HMW PAHs demonstrates its 'plasticity', meeting the catabolic demand of the PAH degraders.

Bacterial benz(a)anthracene catabolic networks in contaminated soils and their modulation by other co-occurring HMW-PAHs

Polycyclic aromatic hydrocarbons (PAHs) are major environmental pollutants in a number of point source contaminated sites, where they are found embedded in complex mixtures containing different polyaromatic compounds. The application of bioremediation technologies is often constrained by unpredictable end-point concentrations enriched in recalcitrant high molecular weight (HMW)-PAHs. The aim of this study was to elucidate the microbial populations and potential interactions involved in the biodegradation of benz(a)anthracene (BaA) in PAH-contaminated soils. The combination of DNA stable isotope probing (DNA-SIP) and shotgun metagenomics of 13C-labeled DNA identified a member of the recently described genus Immundisolibacter as the key BaA-degrading population. Analysis of the corresponding metagenome assembled genome (MAG) revealed a highly conserved and unique genetic organization in this genus, including novel aromatic ring-hydroxylating dioxygenases (RHD). The influence of other HMW-PAHs on BaA degradation was ascertained in soil microcosms spiked with BaA and fluoranthene (FT), pyrene (PY) or chrysene (CHY) in binary mixtures. The co-occurrence of PAHs resulted in a significant delay in the removal of PAHs that were more resistant to biodegradation, and this delay was associated with relevant microbial interactions. Members of Immundisolibacter, associated with the biodegradation of BaA and CHY, were outcompeted by Sphingobium and Mycobacterium, triggered by the presence of FT and PY, respectively. Our findings highlight that interacting microbial populations modulate the fate of PAHs during the biodegradation of contaminant mixtures in soils.

Sandaracinobacteroides saxicola sp. nov., a Zeaxanthin-Producing and Halo-Sensitive Bacterium Isolated from Fully Weathered Granitic Soil, and the Diversity of Its ARHDs

A yellow, Gram-stain-negative, aerobic, non-spore-forming, motile, and rod-shaped bacterial strain designated M6T was isolated from fully weathered granitic soil. The strain showing the highest 16S rRNA gene sequence similarity to M6T was Sandaracinobacteroides hominis SZY PN-1T (96.3%), the only species in the genus Sandaracinobacteroides. The average nucleotide identity and digital DNA-DNA hybridization value between these two strains were 72.6% and 18.0% respectively. Growth was inhibited by NaCl (≥0.1% (w/v)). Strain M6T contained C18:1ω7c (33.8%), C14:0 2-OH (16.6%), summed feature 3 (15.8%), and C16:0 (12.6%) as the major fatty acids. The polar lipids profile consisted of phosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid, four unidentified phospholipids, and four unidentified lipids. The genome of strain M6T was 3.4 Mb with 67.7% GC content. Further genomic analysis revealed a biosynthetic gene cluster for zeaxanthin, the production of which was verified by a high-resolution mass spectrum. The existence of multiple genes for aromatic ring-hydroxylating dioxygenases implies the potential ability for organic pollution controlling. The morphological, physiological, chemotaxonomic, and phylogenetic analysis clearly distinguished this strain from its phylogenetic neighbors, thus strain M6T represents a novel species of the genus Sandaracinobacteroides, for which the name Sandaracinobacteroides saxicola sp. nov. is proposed. The type of strain is M6T (=CGMCC 1.19164T=NBRC 115420T).

DARHD: A sequence database for aromatic ring-hydroxylating dioxygenase analysis and primer evaluation

Biodegradation of aromatic compounds is ubiquitous in the environment and important for controlling organic pollutants. Aromatic ring-hydroxylating dioxygenases (ARHDs) are responsible for the first and rate-limiting step of aerobic biodegradation of aromatic compounds. The ARHD α subunit is a good biomarker for studying functional microorganisms in the environment, however their diversity and corresponding primer coverage are unclear, both of which require a comprehensive sequence database for the ARHD α subunit. Here amino acid sequences of the ARHD α subunit were collected, and a total of 103 sequences were selected as seed sequences that were distributed in 72 bacterial genera with 34 gene names. Based on both homolog search and keyword confirmation against the GenBank, a sequence database of ARHD (DARHD) has been established and 6367 highly credible sequences were retrieved. DARHD contained 407 bacterial genera capable of degrading 38 aromatic substrates, and intricate relationships among the gene name, aromatic substrate and microbial taxa were observed. Thereafter, a total of 136 pairs of primers were collected and assessed. Results showed coverages of most published primers were low. Our research provides new insights for understanding the diversity of ARHD α subunit, and gives guidance on the design and application of primers in the future.

Advances in bacterial Rieske non-heme iron ring-hydroxylating dioxygenases that initiate polycyclic aromatic hydrocarbons degradation

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants, which have received widespread attentions due to their carcinogenic and mutagenic toxicity. The microbial degradation of PAHs are usually started from the hydroxylation, followed by dehydrogenation, ring cleavage and step-by-step removal of branched chains, and finally mineralized by the tricarboxylic acid cycle. Rieske type non-heme iron aromatic ring-hydroxylating dioxygenases (RHOs) or cytochrome P450 oxidases are responsible for the conversion of hydrophobic PAHs into hydrophilic derivatives by the ring hydroxylation. The ring hydroxylation is the first step of PAHs degradation and also one of the rate-limiting steps. Here, we review the distribution, substrate specificity, and substrate recognition mechanisms of RHOs, along with some techniques and methods used for the research of RHOs and PAHs.

Genomic analysis of Gordonia polyisoprenivorans strain R9, a highly effective 17 beta-estradiol- and steroid-degrading bacterium

The increasing levels of estrogens and pollution by other steroids pose considerable challenges to the environment. In this study, the genome of Gordonia polyisoprenivorans strain R9, one of the most effective 17 beta-estradiol- and steroid-degrading bacteria, was sequenced and annotated. The circular chromosome of G. polyisoprenivorans R9 was 6,033,879 bp in size, with an average GC content of 66.91%. More so, 5213 putative protein-coding sequences, 9 rRNA, 49 tRNA, and 3 sRNA genes were predicted. The core-pan gene evolutionary tree for the genus Gordonia showed that G. polyisoprenivorans R9 is clustered with G. polyisoprenivorans VH2 and G. polyisoprenivorans C, with 93.75% and 93.8% similarity to these two strains, respectively. Altogether, the three G. polyisoprenivorans strains contained 3890 core gene clusters. Strain R9 contained 785 specific gene clusters, while 501 and 474 specific gene clusters were identified in strains VH2 and C, respectively. Furthermore, whole genome analysis revealed the existence of the steroids and estrogens degradation pathway in the core genome of all three G. polyisoprenivorans strains, although the G. polyisoprenivorans R9 genome contained more specific estrogen and steroid degradation genes. In strain R9, 207 ABC transporters, 95 short-chain dehydrogenases (SDRs), 26 monooxygenases, 21 dioxygenases, 7 aromatic ring-hydroxylating dioxygenases, and 3 CoA esters were identified, and these are very important for estrogen and steroid transport, and degradation. The results of this study could enhance our understanding of the role of G. polyisoprenivorans R9 in estradiol and steroid degradation as well as evolution within the G. polyisoprenivorans species.

Biphenyl 2,3-Dioxygenase in Pseudomonas alcaliphila JAB1 Is Both Induced by Phenolics and Monoterpenes and Involved in Their Transformation.

The involvement of bacterial aromatic ring-hydroxylating dioxygenases (ARHDs) in the degradation of aromatic pollutants, such as polychlorinated biphenyls (PCBs), has been well studied. However, there is considerable speculation as to the origin of this ability. One hypothesis is centered on a connection between the ability to degrade aromatic pollutants and the necessity of soil bacteria to cope with and/or utilize secondary plant metabolites (SPMs). To investigate this connection, we researched the involvement of biphenyl 2,3-dioxygenase (BPDO), an ARHD essential for the degradation of PCBs, in the metabolism of SPMs in the soil bacterium Pseudomonas alcaliphila JAB1, a versatile degrader of PCBs. We demonstrated the ability of the strain JAB1 to transform a variety of SPMs, namely the flavonoids apigenin, flavone, flavanone, naringenin, fisetin, quercetin, morin, and catechin, caffeic acid, trans-cinnamic acid, and the monoterpenes (S)-limonene and (R)-carvone. Of those, the transformation of flavone, flavanone, and (S)-limonene was conditioned by the activity of JAB1-borne BPDO and thus was researched in more detail, and we found evidence for the limonene monooxygenase activity of the BPDO. Furthermore, the bphA gene in the strain JAB1 was demonstrated to be induced by a wide range of SPMs, with monoterpenes being the strongest inducers of the SPMs tested. Thus, our findings contribute to the growing body of evidence that ARHDs not only play a role in the catabolism of aromatic pollutants, but also of natural plant-derived aromatics, and this study supports the hypothesis that ARHDs participate in ecological processes mediated by SPMs.

Metatranscriptomic Analysis of Oil-Exposed Seawater Bacterial Communities Archived by an Environmental Sample Processor (ESP).

The use of natural marine bacteria as “oil sensors” for the detection of pollution events can be suggested as a novel way of monitoring oil occurrence at sea. Nucleic acid-based devices generically called genosensors are emerging as potentially promising tools for in situ detection of specific microbial marker genes suited for that purpose. Functional marker genes are particularly interesting as targets for oil-related genosensing but their identification remains a challenge. Here, seawater samples, collected in tanks with oil addition mimicking a realistic oil spill scenario, were filtered and archived by the Environmental Sample Processor (ESP), a fully robotized genosensor, and the samples were then used for post-retrieval metatranscriptomic analysis. After extraction, RNA from ESP-archived samples at start, Day 4 and Day 7 of the experiment was used for sequencing. Metatranscriptomics revealed that several KEGG pathways were significantly enriched in samples exposed to oil. However, these pathways were highly expressed also in the non-oil-exposed water samples, most likely as a result of the release of natural organic matter from decaying phytoplankton. Temporary peaks of aliphatic alcohol and aldehyde dehydrogenases and monoaromatic ring-degrading enzymes (e.g., ben, box, and dmp clusters) were observed on Day 4 in both control and oil-exposed and non-exposed tanks. Few alkane 1-monooxygenase genes were upregulated on oil, mostly transcribed by families Porticoccaceae and Rhodobacteraceae, together with aromatic ring-hydroxylating dioxygenases, mostly transcribed by Rhodobacteraceae. Few transcripts from obligate hydrocarbonoclastic genera of Alcanivorax, Oleispira and Cycloclasticus were significantly enriched in the oil-treated exposed tank in comparison to control the non-exposed tank, and these were mostly transporters and genes involved in nitrogen and phosphorous acquisition. This study highlights the importance of seasonality, i.e., phytoplankton occurrence and senescence leading to organic compound release which can be used preferentially by bacteria over oil compounds, delaying the latter process. As a result, such seasonal effect can reduce the sensitivity of genosensing tools employing bacterial functional genes to sense oil. A better understanding of the use of natural organic matter by bacteria involved in oil-biodegradation is needed to develop an array of functional markers enabling the rapid and specific in situ detection of anthropogenic pollution.

Diversity of the Aromatic-Ring-Hydroxylating Dioxygenases in the Monoaromatic Hydrocarbon Degraders Held by a Common Ancestor.

Aromatic ring hydroxylating dioxygenases (ARHDs), harboured by a variety of bacteria, catalyze the initial reaction in the degradation of a wide range of toxic environmental contaminants like aromatic and polycyclic aromatic hydrocarbons (PAHs). Regardless of the source, bacteria harbouring RHDs play major role in the removal of these toxic contaminants. The diversity of ARHDs in contaminated sites is supposed to be huge. However, most of the ARHD diversity studies are based on the PAH degraders and the ARHD diversity in the monoaromatic hydrocarbon degraders has not fully explored yet. In this study, therefore, the ARHD gene from nine different genara of the monoaromatic hydrocarbon degraders including Raoultella, Stenotrophomons, Staphylococcus, Acinetobacter, Pseudomonas, Serratia, Comamonas, Pantoea, and Micrococcus was analysed through polymerase chain reactions and sequencing. The sequence alignments of the ARHD amplicons with 81%-99% homologies were found to be highly related and held by divergent evolution from a common ancestor.

Natural Attenuation Potential of Polychlorinated Biphenyl-Polluted Marine Sediments

The marine environment in Kuwait is polluted with various hazardous chemicals of industrial origin. These include petroleum hydrocarbons, halogenated compounds and heavy metals. Bioremediation with dedicated microorganisms can be effectively applied for reclamation of the polluted marine sediments. However, information on the autochthonous microbes and their ecophysiology is largely lacking. We analyzed sediments from Shuwaikh harbor to detect polychlorinated biphenyls (PCBs) and total petroleum hydrocarbons (TPHs). Then we adopted both culture-dependent and culture-independent (PCR-DGGE) approaches to identify bacterial inhabitants of the polluted marine sediments from Shuwaikh harbor. The chemical analysis revealed spatial variation among the sampling stations in terms of total amount of PCBs, TPHs and the PCB congener fingerprints. Moreover, in all analyzed sediments, the medium-chlorine PCB congeners were more abundant than the low-chlorine and high-chlorine counterparts. PCR-DGGE showed the presence of members of the Proteobacteria, Spirochaetes, Firmicutes and Bacteroidetes in the analyzed sediments. However, Chloroflexi-related bacteria dominated the detected bacterial community. We also enriched a biphenyl-utilizing mixed culture using the W2 station sediment as an inoculum in chemically defined medium using biphenyl as a sole carbon and energy source. The enriched mixed culture consisted mainly of the Firmicute Paenibacillus spp. Sequences of genes encoding putative aromatic ring-hydroxylating dioxygenases were detected in sediments from most sampling stations and the enriched mixed culture. The results suggest the potential of bioremediation as a means for natural attenuation of Shuwaikh harbor sediments polluted with PCBs and TPHs.

Diversity of aromatic hydroxylating dioxygenase genes in mangrove microbiome and their biogeographic patterns across global sites.

Aromatic hydrocarbons (AH), such as polycyclic aromatic hydrocarbons, are compounds largely found in nature. Aromatic‐ring‐hydroxylating dioxygenases (ARHD) are proteins involved in AH degradation pathways. We used ARHD functional genes from an oil‐impacted mangrove area and compared their diversity with other sites around the world to understand the ARHD biogeographic distribution patterns. For this, a comprehensive database was established with 166 operational protein families (OPFs) from 1,758 gene sequences obtained from 15 different sites worldwide, of which twelve are already published studies and three are unpublished. Based on a deduced ARHD peptide sequences consensus phylogeny, we examined trends and divergences in the sequence phylogenetic clustering from the different sites. The taxonomic affiliation of the OPF revealed that Pseudomonas, Streptomyces, Variovorax, Bordetella and Rhodococcus were the five most abundant genera, considering all sites. The functional diversity analysis showed the enzymatic prevalence of benzene 1,2‐dioxygenase, 3‐phenylpropionate dioxygenase and naphthalene 1,2‐dioxygenase, in addition to 10.98% of undefined category ARHDs. The ARHD gene correlation analysis among different sites was essentially important to gain insights on spatial distribution patterns, genetic congruence and ecological coherence of the bacterial groups found. This work revealed the genetic potential from the mangrove sediment for AH biodegradation and a considerable evolutionary proximity among the dioxygenase OPFs found in Antarctica and South America sites, in addition to high level of endemism in each continental region.

Comparative metagenomic analysis of PAH degradation in soil by a mixed microbial consortium

In this study, we used a taxonomic and functional metagenomic approach to analyze some of the effects (e.g. displacement, permanence, disappearance) produced between native microbiota and a previously constructed Polycyclic Aromatic Hydrocarbon (PAH)-degrading microbial consortium during the bioremediation process of a soil polluted with PAHs. Bioaugmentation with a fungal-bacterial consortium and biostimulation of native microbiota using corn stover as texturizer produced appreciable changes in the microbial diversity of polluted soils, shifting native microbial communities in favor of degrading specific populations. Functional metagenomics showed changes in gene abundance suggesting a bias towards aromatic hydrocarbon and intermediary degradation pathways, which greatly favored PAH mineralization. In contrast, pathways favoring the formation of toxic intermediates such as cytochrome P450-mediated reactions were found to be significantly reduced in bioaugmented soils. PAH biodegradation in soil using the microbial consortium was faster and reached higher degradation values (84% after 30 d) as a result of an increased co-metabolic degradation when compared with other mixed microbial consortia. The main differences between inoculated and non-inoculated soils were observed in aromatic ring-hydroxylating dioxygenases, laccase, protocatechuate, salicylate and benzoate-degrading enzyme genes. Based on our results, we propose that several concurrent metabolic pathways are taking place in soils during PAH degradation.

English

‘Conserved hypothetical’ proteins pose a challenge not just for functional genomics, but also to biology in general. As long as there are hundreds of conserved proteins with unknown function in model organisms such as Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae, any discussion towards a ‘complete’ understanding of these biological systems will remain a wishful thinking. Insilico approaches exhibit great promise towards attempts that enable appreciating the plausible roles of these hypothetical proteins. Among the majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multi-domain proteins, created as a result of gene duplication events. Aromatic ring-hydroxylating dioxygenases, also called Rieske dioxygenases (RDOs), are class of multi-domain proteins that catalyze the initial step in microbial aerobic degradation of many aromatic compounds. Investigations here address the computational characterization of hypothetical proteins containing Ferredoxin and Flavodoxin signatures. Consensus sequence of each class of oxidoreductase was obtained by a phylogenetic analysis, involving clustering methods based on evolutionary relationship. A synthetic sequence was developed by combining the consensus, which was used as the basis to search for their homologs via BLAST. The exercise yielded 129 multidomain hypothetical proteins containing both 2Fe-2S (Ferredoxin) and FNR (Flavodoxin) domains. In the current study, 17 proteins with N-terminus FNR domain and C-terminus 2Fe-2S domain are characterized, through homology modelling and docking exercises which suggest dioxygenase activity indicate their plausible roles in degradation of aromatic moieties.

Comparative genomic analysis of six bacteria belonging to the genus Novosphingobium: insights into marine adaptation, cell-cell signaling and bioremediation

BackgroundBacteria belonging to the genus Novosphingobium are known to be metabolically versatile and occupy different ecological niches. In the absence of genomic data and/or analysis, knowledge of the bacteria that belong to this genus is currently limited to biochemical characteristics. In this study, we analyzed the whole genome sequencing data of six bacteria in the Novosphingobium genus and provide evidence to show the presence of genes that are associated with salt tolerance, cell-cell signaling and aromatic compound biodegradation phenotypes. Additionally, we show the taxonomic relationship between the sequenced bacteria based on phylogenomic analysis, average amino acid identity (AAI) and genomic signatures.ResultsThe taxonomic clustering of Novosphingobium strains is generally influenced by their isolation source. AAI and genomic signature provide strong support the classification of Novosphingobium sp. PP1Y as Novosphingobium pentaromaticivorans PP1Y. The identification and subsequent functional annotation of the unique core genome in the marine Novosphingobium bacteria show that ectoine synthesis may be the main contributing factor in salt water adaptation. Genes coding for the synthesis and receptor of the cell-cell signaling molecules, of the N-acyl-homoserine lactones (AHL) class are identified. Notably, a solo luxR homolog was found in strain PP1Y that may have been recently acquired via horizontal gene transfer as evident by the presence of multiple mobile elements upstream of the gene. Additionally, phylogenetic tree analysis and sequence comparison with functionally validated aromatic ring hydroxylating dioxygenases (ARDO) revealed the presence of several ARDOs (oxygenase) in Novosphingobium bacteria with the majority of them belonging to the Groups II and III of the enzyme.ConclusionsThe combination of prior knowledge on the distinctive phenotypes of Novosphingobium strains and meta-analysis of their whole genomes enables the identification of several genes that are relevant in industrial applications and bioremediation. The results from such targeted but comprehensive comparative genomics analysis have the potential to contribute to the understanding of adaptation, cell-cell communication and bioremediation properties of bacteria belonging to the genus Novosphingobium.

Analysis of multi-domain hypothetical proteins containing iron-sulphur clusters and fad ligands reveal rieske dioxygenase activity suggesting their plausible roles in bioremediation.

‘Conserved hypothetical’ proteins pose a challenge not just for functional genomics, but also to biology in general. As long as there are hundreds of conserved proteins with unknown function in model organisms such as Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae, any discussion towards a ‘complete’ understanding of these biological systems will remain a wishful thinking. Insilico approaches exhibit great promise towards attempts that enable appreciating the plausible roles of these hypothetical proteins. Among the majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multi-domain proteins, created as a result of gene duplication events. Aromatic ring-hydroxylating dioxygenases, also called Rieske dioxygenases (RDOs), are class of multi-domain proteins that catalyze the initial step in microbial aerobic degradation of many aromatic compounds. Investigations here address the computational characterization of hypothetical proteins containing Ferredoxin and Flavodoxin signatures. Consensus sequence of each class of oxidoreductase was obtained by a phylogenetic analysis, involving clustering methods based on evolutionary relationship. A synthetic sequence was developed by combining the consensus, which was used as the basis to search for their homologs via BLAST. The exercise yielded 129 multidomain hypothetical proteins containing both 2Fe-2S (Ferredoxin) and FNR (Flavodoxin) domains. In the current study, 40 proteins with N-terminus 2Fe-2S domain and C-terminus FNR domain are characterized, through homology modelling and docking exercises which suggest dioxygenase activity indicating their plausible roles in degradation of aromatic moieties.

Functional diversity of bacterial genes associated with aromatic hydrocarbon degradation in anthropogenic dark earth of Amazonia

The objective of this work was to evaluate the catabolic gene diversity for the bacterial degradation of aromatic hydrocarbons in anthropogenic dark earth of Amazonia (ADE) and their biochar (BC). Functional diversity analyses in ADE soils can provide information on how adaptive microorganisms may influence the fertility of soils and what is their involvement in biogeochemical cycles. For this, clone libraries containing the gene encoding for the alpha subunit of aromatic ring-hydroxylating dioxygenases (α-ARHD bacterial gene) were constructed, totaling 800 clones. These libraries were prepared from samples of an ADE soil under two different land uses, located at the Caldeirão Experimental Station - secondary forest (SF) and agriculture (AG) -, and the biochar (SF_BC and AG_BC, respectively). Heterogeneity estimates indicated greater diversity in BC libraries; and Venn diagrams showed more unique operational protein clusters (OPC) in the SF_BC library than the ADE soil, which indicates that specific metabolic processes may occur in biochar. Phylogenetic analysis showed unidentified dioxygenases in ADE soils. Libraries containing functional gene encoding for the alpha subunit of the aromatic ring-hydroxylating dioxygenases (ARHD) gene from biochar show higher diversity indices than those of ADE under secondary forest and agriculture.

Multiplicity of genes for aromatic ring-hydroxylating dioxygenases in Mycobacterium isolate KMS and their regulation

Mycobacterium sp. strain KMS has bioremediation potential for polycyclic aromatic hydrocarbons (PAHs), such as pyrene, and smaller ring aromatics, such as benzoate. Degradation of these aromatics involves oxidation catalyzed by aromatic ring-hydroxylating dioxygenases. Multiple genes encoding dioxygenases exist in KMS: ten genes encode large-subunits with homology to phenylpropionate dioxygenase genes, sixteen pairs of adjacent genes encode alpha- and beta-subunits of dioxygenase and two genes encode beta-subunits. These genes include orthologs of nid genes essential for degradation of multi-ring PAHs in M. vanbaalenii isolate PYR-1. The multiplicity of genes in part is explained by block duplication that results in two or three copies of certain genes on the chromosome, a linear plasmid, and a circular plasmid within the KMS genome. Quantitative real-time PCR showed that four dioxygenase beta-subunit nid genes from operons with almost identical promoter sequences otherwise unique in the genome were induced by pyrene to similar extents. No induction occurred with benzoate. Unlike isolate PYR-1, isolate KMS has an operon specifying benzoate catabolism and the expression of the alpha-subunit dioxygenase gene was activated by benzoate but not pyrene. These studies showed that isolate KMS had a genome well adapted to utilization of different aromatic compounds.

Novel Biphenyl-Oxidizing Bacteria and Dioxygenase Genes from a Korean Tidal Mudflat

Gene-targeted FLX titanium pyrosequencing integrated with stable isotope probing (SIP) using [(13)C]biphenyl substrate revealed that tidal mudflat sediments harbor novel aromatic ring hydroxylating dioxygenases (ARHD). More than 80% of the detected ARHD genes comprise four clades (0.5 distance) with 49 to 70% amino acid identity to sequences in public databases. The 16S rRNA sequences enriched in the (13)C fraction were from the Betaproteobacteria, bacilli (primarily Paenibacillus-like), and unclassified phyla.